Use of avian antibodies

ABSTRACT

The present invention relates to use of avian antibodies and/or antigen binding fragments thereof, for the production of a drug for treatment and/or prevention of respiratory tract infection. The drug is administered through local application at the oral cavity and/or pharynx.

The present application is the national stage under 35 U.S.C. 371 ofPCT/SE98/00526, filed Mar. 20, 1998, which claims priority from Swedishapplication 9701026-8, filed Mar. 20, 1997.

TECHNICAL FIELD

The present invention relates to the use of avian antibodies againstinfectious antigen for treatment and/or prevention of respiratory tractinfections, The antibodies are administered through local application atthe oral cavity, and/or pharynx,

BACKGROUND OF THE INVENTION AND PRIOR ART

It is well known that domestic avian species, e.g. hens, produce hightitres of antibodies in their eggs against factors against which theyhave been immunised (4), In several trials avian antibodies have beenused to prevent or treat bacterial or viral infections in the digestivetract of different animals with promising results (12-17), but there isno evidence in the scientific literature that avian antibodies, orantibodies of any other origin, have been used in preventing or treatingrespiratory tract infections, neither in mammals nor humans.Traditionally respiratory tract infections are treated with conventionaltherapy, such as antibiotic treatment. The reason for not usingantibodies is that no possible way of administering the antibodies, whentreating respiratory tract infections, has been seen. Most surprisingly,the present inventors found that the administration of antibodiesthrough local application at the oral cavity and/or pharynx could beused to treat respiratory tract infections.

SUMMARY OF THE INVENTION

The present invention suggests, for the first time, use of avianantibodies and/or antigen binding fragments thereof, for the productionof a drug for preventing and/or treating respiratory tract infections.

Most surprisingly, the present invention suggests, for the first time,that avian antibodies can be used to prevent and/or treat respiratorytract infections when administered through local application at the oralcavity and/or pharynx, preferably by gargling and/or swallowing. Thepresent inventors have verified this by clinical studies on humans.

The present invention relates to treatment of respiratory tractinfections—i.e. infections in the nasal cavity, paranasal sinuses thelymphatic ring in the oropharynx, larinx, trachea, bronchi, bronchioliand/or alveoli, i.e. all the way down the respiratory tree—caused bye.g. Pseudomonads and/or related microorganisms.

Treatment and/or prevention of respiratory tract infections according tothe invention are particularly appropriate for those individuals havingincreased susceptibility to infections or increased risk of catchinginfections, since the treatment is mild and not accompanied byundesirable side effects.

DETAILED DESCRIPTION OF THE INVENTION

To test the use of antibodies for treating respiratory tract infectionsthe present inventors choose the group of patients suffering from cysticfibrosis (CF). These patients have increased susceptibility toinfections with reoccurring or chronic respiratory tract infections ofPseudomonas aeruginosa. Cystic fibrosis patients have hitherto had torely on conventional therapy, such as antibiotic treatment which issometimes not successful and accompanied by undesired side effects.

Chronic colonisation with Pseudomonas aeruginosa in the respiratorytract of patients with cystic fibrosis (CF) is a principal cause of thehigh morbidity and mortality in this disease. It is very difficult toget rid of Pseudomonas aeruginosa once it has been isolated from thesputum of CF-patients. Only a temporary eradication of this pathogen canbe achieved by vigorous antibiotic treatment during very early phases ofcolonization—and the bacteria will return very soon (1-3). The bestresults hitherto have been reported from the CF-center in Copenhagen (2)where 14 CF-patients were treated daily with oral ciprofloxacin andinhalation of colistin for 3 weeks at their first Pseudomonasaeruginosa-positive culture. After treatment, these CF-patients wereobserved for up to 27 months (totally 214 months; mean 15.3 months).During this time 2 of the patients became chronically colonized withPseudomonas aeruginosa (defined as six consecutive sputum culturespositive for the bacteria) and there were 49 sputum cultures positivefor Pseudomonas aeruginosa out of 214 (=23%). From another Copenhagenstudy it is reported that Pseudomonas aeruginosa reoccured in sputumcultures already 4 months after a course of anti-pseudomonaschemotherapy in 98.3% of the CF-patients (3).

Each egg from a hen contains more than 100 mg antibodies. Theseantibodies are produced by hens and transported to their eggs where theyare found in high concentrations. High titres of specific antibodiesagainst bacteria were achieved by repeated immunization of hens withkilled specific bacteria or fragments thereof, Specific antibodiesagainst Pseudomonas aeruginosa have been produced by repeatedimmunizations of hens with killed Pseudomonas aeruginosa. Eggs fromthese hens have been used to make a solution with high specific antibodyconcentration.

An antibody consists of two parts; a Fab fragment which is the part ofthe antibody that binds to the antigen and a Fc fragment that lackantigen binding properties, The basis of oral administration ofimmunoglobulins has been attributed to interference with bacterialadherence and neutralization of toxins produced by the pathogens. Theprotective effect should therefore be conferred by either the whole IgYmolecule or the antigen binding fragment thereof since both are capableof above functions. In fact this was demonstrated by the work of Ahrenand Svennerholm (5) who observed that Fab fragments of anti-CFA/Ireduced fluid secretion (diarrhoea) almost as effectively as non-cleavedimmunoglobulin fraction of the serum. Fab fragments from chicken IgYwill thus have similar protective functions against bacterial infectionsas the whole IgY molecule.

In contrast to antibodies from mammals (eg bovine), the antibodies fromeggs (6) do not activate the human complement system (7). This is atremendous advantage since activated complement factors are veryeffective mediators of inflammatory reactions. In addition, antibodiesfrom eggs do not react with rheumafactors (8), human Fc-receptors (9),bacterial Fc-receptors (10) or human anti-mouse IgG-antibodies (11),which make them very safe to use.

The following examples are provided to further illustrate the inventionwithout being limiting.

EXAMPLE 1

Preparation of avian antibodies against Pseudomonas aeruginosa.Pseudomonas aeruginosa bacteria were killed by formaldehyde fixation.The killed bacteria were washed in 0.9% NaCl and frozen at −20° C. 0.5mL of bacteria was mixed with 0.5 mL of Freunds incomplete adjuvant andused for intramuscular immunization of domestic hens. The hens wereimmunized four times with 2 weeks between the immunizations. The eggswere collected after the initial immunization period of eight weeks andthen continuously. The hens recieved further booster immunizations with2-3 months interval so that they would remain in hyperimmunizedcondition. The antibodies were purified by the water dilution methodaccording to Akita and Nakai, 1993 (18). After the precipitate had beenremoved by centrifugation the supernatant was frozen at minus 20° C. in30-70 mL portions. The antibody titre of the final preparations werechecked by immunoblotting.

EXAMPLE II Case Reports

At their first colonization with Pseudomonas aeruginosa in therespiratory tract, two CF-patients were treated with the oral antibioticciprofloxacin and antibiotic inhalations of colistin or tobramycin forthree weeks. Simultaneously with the antibiotic treatment, they startedto gargle daily (for 2 minutes) with the solution according to example 1and thereafter to swallow specific antibodies against Pseudomonasaeruginosa. After this three week period, the patients have continouslyreceived antibodies according to the above procedure, but withoutantibiotics, for together more than 30 months (26 and 4 months,respectively). All sputum cultures during the treatment period have beennegative for Pseudomonas aeruginosa. (0 positive out of 28=0%; thisshould be compared with the results from the Copenhagen study, referredto above, which was 49 out of 214=23% p<0.005). Thus, a completeeradication of Pseudomonas aeruginosa in the respiratory tract ofCF-patients is achieved. The results strongly suggest that the treatmentwith gargling and swallowing of antibodies against Pseudomonasaeruginosa according to the invention is effective to prevent chroniccolonization of the bacteria. The effect is with all probability due tothe local effect of-the avian antibodies in the oral cavity and/orpharynx, since the antibodies, when swallowed, are degraded byintestinal proteolytic enzymes and are not bloodborne.

For these patients, the dose is preferably about 50 mg IgY per day.Precautions have been made to avoid bacterial contamination of thesolutions, which thereafter have been kept at −20° C. The CF-patientshave been asked to take out a bottle with 30-70 ml of the solution fromthe freezer each morning and to gargle with this solution in the eveningfor 2 minutes and thereafter to swallow it.

Case 1. A 21 year old female with CF. Diagnosed at birth by screeningwith albumin in meconium (179 mg albumin/g dry weight meconium) andsubsequent sweat test (Na 101, Cl 122 mmol/kg sweat). Already during herfirst three weeks of life there were severe feeding problems. She hadfrequent stools and was vomiting considerably and her weight went downfrom 3430 g to 2930 g despite a vigorous appetite. After proper therapy(pancreatic enzymes, breast feeding, extra vitamins, mist tent, physicaltherapy and anti-staphyloccal antibiotics at every respiratoryinfection) was instituted she started to gain weight and was doing quitewell. Her height was steadily slightly above the mean for her age to afinal height of 169 cm; her weight was usually at the mean for her agebut had a severe dip at 7-8 years of age and she did not come back tomean weight until 15 years of age. During the last two years her weighthas been around 53 kg, ie BMI (body mass index) 18,6. From 7½ years ofage she has always had Staphylococcus aureus in her sputum andoccasionally also Hemophilus influenzae or Proteus mirabilis. Her chestX-rays showed a slight but steady progress of typical CF-changes and herlung function deteriorated slowly to FVC (forced vital capacity) ofabout 75%, and FEV1 (forced expiratory volume in one second) to about50% of predicted values at the age of 19 years. Her first colonizationwith Pseudomonas aeruginosa occurred in November 1989. This waseffectively treated with azactam and gentamycin iv in two periods offourteen days each and addition of ciprofloxain orally at the secondcourse. Thereafter her sputum cultures returned to the earlier patternof Staphylococcus aureus and Hemophilus influenzae until August 1994. Atthis time the bacteria were eliminated after a 14 days course ofceftazidim and tobramycin iv.

However, already in January 1995, Pseudomonas aeruginosa was again foundin her sputum. At this time it was decided to give her an antibioticcourse according to the Copenhagen model (2) with ciprofloxin 750 mg×3orally and colistin 2 million U×2 by inhalation for three weeks and atthe same time start with daily gargling and swallowing of avianantibodies against Pseudomonas aeruginosa. The daily intake ofantibodies has continued since then (now for 26 months). There has beenno new appearence of Pseudomonas aeruginosa and the pattern of bacteriain her sputum has returned to the usual. She has never had anyprecipitins or antibodies against Psedomonas aeruginosa in her serum. Noside effects of the treatment have been seen in her blood (red and whiteblood cells, trombocytes, liver enzymes or creatinine values). Her chestX-rays have been essentially unchanged since 1995 as well as her lungfunction tests (FVC still 75% and FEV1 now 45% of predicted). She is anextremely active woman and her working capacity (150 Watt) must beconsidered very good in regard to her severe disease.

Case 2. A 17 year old girl with CF During her first two years of lifeshe had recurrent obstructive bronchitis, frequent greasy, foulsmelling, loose stools and poor weight gain (weight 9,5 kg at 2 years ofage). At this time, sweat tests were performed and revelad the diagnosisof CF. Intensive treatment was then instituted. Her weight came back tomean values but her height followed the −2 SD curve and has stopped at151 cm. Her main problem has been recurrent stomach ache of the type“distal intestinal obstruction syndrome (DIOS)”. Her lungs have alwaysbeen very good—chest X-rays show only minimal changes, lung functiontests have all been at or above mean for her age and height (theexception is RV (residual volume): 1,14=186% of predicted). Her workingcapacity (120 Watt) is nearly normal. Her sputum cultures had alwaysshown Staphylococcus aureus and/or Hemophilus influenzae until November1996 at which time the cultures for the first time showed Pseudomonasaeruginosa. She then immediately got a three weeks course ofciprofloxacin 750 mg×2 orally and tobramycin 320 mg×2 by inhalation(colistin was not available). Simultaneously she started daily garglingand swallowing of avian antibodies against Pseudomonas aeruginosa withwhich she has continued since then, Pseudomonas was eradicated and sincethen (now for 4 months) she has had her usual bacteria in her sputum.She continues to do well.

Equally good results have been achieved for another nine patients andthe results strongly suggest a prophylactic and therapeutic effect ofthe treatment of respiratory tract infection of Pseudomonas aeruginosawith avian antibodies against the same according to the invention. In 30sputum cultures during the therapeutic time period of the two patientsdescribed above no Pseudomonas aeruginosa positive cultures have beenfound. In contrast, in the study from Copenhagen referred to above,there were 23% positive cultures, and as many as 75-100% of thesepatients had at least one positive culture during a similar observationperiod as in the present invention.

REFERENCES

1. Høiby N., Pseudomonas infection in Cystic fibrosis, In CysticFibrosis, Current topics, Vol. 1, edited by Dodge J. A., Brock D. J. H.& Widdicombe J. H., John Wiley & Sons Ltd., London, 1993, pp 251-268.

2. Valerius N. H., Koch C., Høiby N., Prevention of chronic Pseudomonasaeruginosa colonisation in cystic fibrosis by early treatment; TheLancel 1991;338; 725-726

3. Szaff M., Høiby N. and Flensborg E. W.; Frequent antibiotic therapyimproves survival of cystic fibrosis patients with chronic Pseudomonasaeruginosa infection; Acta Paediatr Scand 72:651-657, 1983.

4. Larsson A., B{dot over (a)}löw R. -M., Lindahl T. L. and Forsberg P.-O. (1993) Chicken IgG: Utilizing the evolutionary advantage; PoultryScience, 72, 1807-1812.

5. Ahren C M., Svennerholm A M. (1982) Synergistic protective effect ofantibodies against E. coli enterotoxin and colonization factor antigens.Infec. Immun. 28, 74,

6. Larsson A, and Lindahl T. L. (1993) Chicken antibodies: A tool toavoid interference in immunological assays; Avian immunology inprogress, 62, 97-102.

7. Larsson A., Wej{dot over (a)}ker P. -E., Forsberg P. O. and LindahlT. L. (1992) Chicken antibodies: A tool to avoid interference bycomplement activation in ELISA; J. Immunol. Methods (1992) 156, 79-83.

8. Larsson A. and Sjöquist J. (1988) Chicken antibodies: A tool to avoidfalse positive results by rheumatoid factor in latex fixation tests; J.Immunol. Methods 108, 205-208.

9. Lindahl T. L., Festin R. and Larsson A. (1992), Studies of fibrinogenbinding to platelets by flow cytometry: An improved method for detectionof platelet activation; Thrombosis and Haemostasis, 68, 221-225.

10. Larsson A. and Lindahl T. L. (1993), Chicken anti-protein G for thedetection of small amounts of protein G.; Hybridoma, 12, 143-147.

11. Larsson A. and Mellstedt H. (1992), Chicken antibodies: a tool toavoid interference by human anti-mouse antibodies in ELISA after in vivotreatment with murine monoclonal antibodies; Hybridoma 11, 33-39.

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13. Ebina T., Tsukada K., Umezu K., Nose M., Tsuda K., Hatta H., Kim M.and Yamamoto T., Gastroenteritis in suckling mice caused by humanrotavirus can be prevented with egg yolk immunoglobulin (IgY) andtreated with a protein-bound polysaccharide preparation (PSK);Microbiol. Immunol., 34; (1990) 617.

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17. European Patent Office publication number 225 254 B1

18. Akita, E M and Nakai, S. (1993) Comparison of four purificationmethods for the production of immunoglobulins from eggs laid by hensimmunized with an enterotoxigenic E. Coli strain. J. Immunol. Methods,60, 207-214

What is claimed is:
 1. A method for preventing bacterial infection in apatient suffering from cystic fibrosis, who is not currently sufferingfrom a bacterial infection of the lungs and bronchi, comprising:administering to said patient an amount of avian antibodies againstantigens of said bacterial infection sufficient to prevent bacterialinfection, wherein said administration is at the oral cavity and pharynxof the patient.
 2. The method according to claim 1 wherein the avianantibodies are antibodies against Pseudomonas.
 3. The method accordingto claim 2 wherein said avian antibodies are in a solution and saidsolution is administered by at least one of gargling and swallowing. 4.The method according to claim 3 wherein said solution is administered bygargling and then optionally swallowing.
 5. The method according toclaim 1 wherein said avian antibodies are in a solution and saidsolution is administered by at least one of gargling and swallowing. 6.The method according to claim 5 wherein said solution is administered bygargling and then optionally swallowing.